In the adult brain, sensory stimulation leads to a localized increase in blood flow in a particular region of the brain. This blood flow increase causes a decrease in deoxy-hemoglobin that is detected by functional magnetic resonance imaging (fMRI) as the blood oxygen level dependent (BOLD) signal. However, prior studies in infants and children have revealed widely varying and sometimes inverted hemodynamic responses in the developing brain. Our preliminary data suggest that these differences are due, at least in part, to the immaturity of neurovascular coupling in the neonatal brain, potentially confounding interpretation of fMRI in young subjects. With fMRI increasingly being used for studies of brain development, and to understand developmental disorders such as autism and attention deficit disorder, an improved understanding of vascular control in the neonatal brain is urgently needed. This project will use in-vivo optical imaging and microscopy techniques to study the relationship between neuronal activity and blood flow in the neonatal rodent brain. We have already completed preliminary studies characterizing the evolution of the hemodynamic response to somatosensory stimulus in neonatal rat pups. This work confirmed the presence of 'inverted' responses in neonatal rats, and charted the progression of the response between postnatal days 12 - 23 via an intermediate bi-phasic response in which localized initial hyperemia gradually increased until the response was fully positive. Additionally, a strong vasoconstriction component was found to be present from birth, and clear signs of immature cerebral autoregulation were observed. To complete my dissertation, I propose to build upon this work to characterize the developing response at a cellular and vascular level through the use of both exposed-cortex optical imaging and in-vivo two-photon microscopy (aim 1). The goal of this aim will be to elucidate key components of the neurovascular unit by observing their assembly alongside maturation of functional hyperemia. I also plan to study the degree to which the neonatal hemodynamic response represents underlying neuronal activity using wide-field calcium sensitive dye imaging, electrophysiology, and optogenetics (aim 2). The goal of this aim will be to bring clarity to the interpretation of functional imaging results in infants and childre. The combination of wide-field optical imaging, in-vivo two-photon microscopy, and optogenetics that will be used in this study provides a unique approach to the examination of neonatal neurovascular coupling. We expect that the results of this work will have a significant impact on the field of neonatal brain imaging (both clinically and experimentally). Our results may also provide important insights into normal adult neurovascular coupling, and could potentially identify new biomarkers for normal and abnormal development of the brain's cerebrovascular, metabolic, and autoregulatory systems.